This invention relates to improved methods for generating phosphorylatable polypeptides, polypeptides generated using those methods, DNA sequences encoding those polypeptides, and their use in diagnosis and treatment of cancer and other diseases.
Labeled polypeptides are used in a variety of applications. For instance, labeled monoclonal antibodies (MAbs) have been widely used in radio-immunotherapy, diagnostic imaging and staging of tumors.
Labeled monoclonal antibodies (MAbs) have great applicability for the diagnosis and treatment of cancer for several reasons. First, most tumor populations express tumor antigens in a heterogeneous pattern. Some of the cells in the population will not be expressing the target tumor antigen and therefore will not be recognized by the monoclonal antibody. With the use of MAbs to deliver drugs or toxins to tumor cells, the cells which lack the tumor antigen remain untouched. In contrast, radio labeled MAbs provide the advantage of destroying cells within a radius of a few cell diameters around the tumor cell to which the MAb binds. It has been shown that an 131I-labeled MAb can deliver a therapeutic dose of radiation to antigen negative cells. Second, in the case of carcinomas, the tumor antigens are stable on the cell surface and are not internalized. For a drug or toxin to be effective, it is necessary to have it enter the cell. In contrast, radio labeled MAbs kill the tumor cells after binding to the surface and do not require entry into the cell. Therefore, this technique has applicability to great variety of cancers. Furthermore, the use of interferons and other cytokines can be used to enhance the expression of tumor associated antigens on cells providing a better target for monoclonal antibodies and minimize or even eliminate tumor cells previously not expressing the tumor antigen.
In radio-immunotherapy, 131I has been commonly used for cancer therapy. However since iodine labeling is not site specific, it results in a heterogeneous population of labeled MAbs with various affinities for antigen and significant inactivation of the Mab. Iodine-labeled polypeptides can also undergo dehalogenation, which can eliminate 131I from tumors before it starts to function. Another disadvantage of iodine labeling is that iodine can concentrate in the thyroid, salivary glands and stomach, which can pose health problems for patients and health care personnel.
Compared to 131I, 32P has been considered to be a better option for radio-immunotherapy. Being a pure β-emitter, it has high energy (Emax 1700 keV, compared to 131I, 182 keV) which is strong enough for cancer therapy. However the utilization of this radioisotope was greatly limited due to the difficulties in 32P labeling of MAbs. A 32P labeled peptide can also be chemically coupled to the polypeptide via lysine residues. However, the peptide-Ab conjugation is not site specific, which, like iodine labeling, can also compromise the Ag binding ability of the MAb.
This 32P labeling problem was not satisfactorily solved until the development of a simple and rapid labeling procedure and the construction of a phosphorylatable fusion polypeptide by the introduction of a peptide kinase recognition site into the polypeptide. See, for example, U.S. Pat. No. 5,986,061, the disclosure of which is incorporated by reference herein in its entirety. This is a simple, efficient way to label polypeptides using radio-nucleotides, and is applicable to virtually any polypeptide. Many polypeptide kinase recognition sites can be introduced into polypeptides and serve as useful tags for a variety of purposes. The introduction of polypeptide kinase recognition sites into polypeptides can be achieved without modifying the essential structure or function of the polypeptides. Because polypeptides modified by these procedures retain their activity after phosphorylation, they can be used in many applications.
Phosphorylatable MAbs (MAb-chB72.3-P, MAb-chCC49K1, MAb-chCC49CKI, MAb-chCC49CKII and MAb-chCC49Tyr) can be created by inserting the predicted consensus sequences for phosphorylation by the cAMP-dependent polypeptide kinase and other polypeptide kinases, such as casein kinase I, casein kinase II and the Src tyrosine kinase, at the carboxyl terminus of the heavy chain constant region of MAb-chB72.3-P or MAb-chCC49. These MAbs are purified and phosphorylated by the appropriate polypeptide kinase with [γ-32P]ATP to high specific activity. These [32P]MAbs bind to cells expressing TAG-72 antigens with high specificity. In all these cases, the phosphate is stable in vitro in various sera so that less than 8% of the phosphate is hydrolyzed in 24 hours.
However, it has been found that the attached 32P in the above phosphorylatable antibodies is not sufficiently stable in buffer or serum to be useful for in vivo applications in animals and humans. Several methods have been suggested to improve the stabilities of the phosphorylatable MAbs. Since RRX(S/T) is a PKA recognition site, changing the amino acid residue X or the amino acid residues downstream of this site changes the stability of the phosphorylatable MAbs. It has also been found that using threonine, instead of serine, in the PKA recognition site increases the stability of the phosphorylatable Mabs, although this would compromise the efficiency of the phosphorylation dramatically. Alternatively, the stability of the phosphorylatable MAbs might also be changed if other phosphorylation enzymes are used. There is no assurance that these approaches would be satisfactory.
The choice of putative phosphorylation sites can at times be tricky since many point mutations, insertions or deletions may dramatically change the conformation of the entire molecule or at least render the polypeptide less functional. In addition, those sites might be potentially unaccessible to the intended kinases due to steric hinderance. In the past, these problems were dealt with using such inefficient and time-consuming methods as trial-and-error.
Accordingly, what is needed is a reasonably accurate yet highly efficient means to carry out this process, not only for labeling phosphorylatable monoclonal antibodies, but also as a general method for generating any phosphorylatable polypeptides.